Protection of the 2′-Hydroxy Function of Ribonucleosides as an Iminooxymethyl Propanoate and Its 2′-O-Deprotection through an Intramolecular Decarboxylative Elimination Process

Article abstract of DOI:10.1002/ejoc.201601308

The design and implementation of 2′-hydroxy protecting groups for ribonucleosides is still a daunting challenge to overcome when assembling RNA (ribonucleic acid) sequences for therapeutic applications. The reaction of 2′-O-aminooxymethylribonucleosides with ethyl pyruvate results in the formation of 2′-O-iminooxymethyl ethyl propanoates. The cleavage of this type of 2′-O-protecting groups is demonstrated through saponification of the esters to 2′-O-iminooxymethyl propanoate salts, which, when needed, decarboxylate quantitatively at 55 °C in the presence of tetra-n-butylammonium fluoride or chloride in dimethyl sulfoxide (DMSO) to produce all four native ribonucleosides.

Full text of DOI:10.1002/ejoc.201601308

A Journal of
Accepted Article
Title: Protection of the 2’-Hydroxy Function of Ribonucleosides as an Iminooxyme-
thyl Propanoate and its 2’-O-Deprotection via an Intramolecular Decarboxylati-
ve Elimination Process
Authors: Jacek Cieslak; Andrzej Grajkowski; Cristina Aus´ın; Serge L. Beaucage
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201601308
Link to VoR: http://dx.doi.org/10.1002/ejoc.201601308
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Protection of the 2’-Hydroxy Function of Ribonucleosides as an
Iminooxymethyl Propanoate and its 2’-O-Deprotection via an
Intramolecular Decarboxylative Elimination Process
Jacek Cieślak, Andrzej Grajkowski, Cristina Ausίn, and Serge L. Beaucage*[a]
Abstract: The design and implementation of 2’-hydroxy protecting
groups for ribonucleosides is still a daunting challenge to overcome
when assembling RNA sequences for therapeutic applications. The
reaction of 2’-O-aminooxymethylribonucleosides with ethyl pyruvate
results in the formation of 2’-O-iminooxymethyl propanoic acid ethyl
esters. The deprotection of this type of 2’-O-protecting groups is
demonstrated through saponification of the esters to 2’-O-imino-
oxymethyl propanoate salts, which when needed, decarboxylate
quantitatively at 55 °C in the presence of tetra-n-butylammonium
fluoride or chloride in dimethyl sulfoxide to produce all four native
ribonucleosides.
levulinyloxymethyl,[10a,b]
dichloroacetyl-N-methyl)aminobenzyloxymethyl
pivaloyloxymethyl,[11a,b]
4-(N-
and the 2-
[12a,b]
cyano-2,2-dimethylethanimine-N-oxymethyl[13] groups. The use
of 2’-O-aminooxymethylribonucleosides[13,14] in the synthesis of
the latter 2’-hydroxy protecting group has proven to be a
straightforward, efficient and cost-effective approach to the
chemical synthesis of high quality RNA sequences in the context
of RNA interference applications. However, the fluoride-assisted
deprotection rates of the 2-cyano-2,2-dimethylethanimine-N-
oxymethyl group, in addition to those of any fluoride-sensitive 2’-
hydroxy protecting groups, decrease with increasing RNA chain
length. Electrostatic repulsion between fluoride ions and the
negatively charged phosphodiester functions of RNA sequences
is likely to increase proportionally with the number of
phosphodiesters.[13] With the objective of mitigating this
drawback, we report herein the application of an iminooxymethyl
group for 2’-hydroxy protection of ribonucleosides and
demonstrate its unprecedented cleavage through an innovative,
entropically-driven, intramolecular decarboxylative process.
As shown in Scheme 1, the usefulness of 2’-O-aminooxy-
methylribonucleosides (1a-d) in the development of novel 2’-
hydroxy protective groups is further demonstrated by mixing the
ribonucleoside 1a[13,14] with ethyl pyruvate in the presence of
drops of concd. HCl in MeOH. After 1 h at 25 °C, the 2’-O-
iminooxymethyl propanoic acid ethyl ester derivative 2a was
On the basis of recent developments in RNA interference to
silence the expression of specific genes, the availability of rapid
and efficient methods for the chemical synthesis of RNA
sequences in sufficient quantity and purity for pharmaceutical
applications has become an urgent issue.
challenge in the chemical synthesis of oligoribonucleotides is
designing suitable 2’-hydroxy protecting group for
A formidable
a
ribonucleosides. Indeed, the protecting group should optimally:
(i) be easy to introduce; (ii) remain completely stable throughout
the full assembly of the RNA sequence and particularly under
the conditions used for nucleobase and phosphate deprotection,
and release of the sequence from the solid support; (iii) be
totally removable under conditions that do not compromise the
structural integrity of the RNA sequence.[1,2a-c] The 2’-hydroxy
protecting group should preferably be structurally flexible and
sterically small enough to permit rapid phosphoramidite coupling
kinetics and high coupling efficiencies. In this regard, a number
of acetal, acetalester and orthoester protecting groups have
been investigated with the objective of imparting ribonucleoside
phosphoramidites with coupling kinetics and efficiencies
matching those of deoxyribonucleoside phosphoramidites.
These protecting groups include the: 2-nitrobenzyloxymethyl,[3]
isolated in
a
post-silica gel purification yield of 78%.
Saponification of 2a upon treatment with 0.1 M NaOH produced
the 2’-O-iminooxymethyl propanoate salt 3a in a quantitative
yield. Inspired by reports on the decarboxylation of α-keto
acids,[15a,b] we hypothesized that α-ketoxime acid derivatives
could perhaps be amenable to decarboxylation under mild
conditions. Indeed, heating 3a in the presence of
4-nitrobenzyloxymethyl,[4]
bis(2-acetoxyethoxy)methyl,[5]
triisopropylsilyloxymethyl,[6] 2-cyanoethoxymethyl,[7a,b] 2-tert-
butyldithiomethyl,[8] 2-(4-tolylsulfonyl)ethoxymethyl,[9a,b]
[a]
Dr. J. Cieślak, Dr. A. Grajkowski, Dr. C. Ausίn, Dr. Serge L.
Beaucage
Division of Biotechnology Review and Research IV, Center for Drug
Evaluation and Research, Food and Drug Administration,
10903 New Hampshire Avenue, Silver Spring, MD 20933 (USA)
E-mail: serge.beaucage@fda.hhs.gov
Scheme 1. Implementation of a 2’-O-iminooxymethyl propanoate protecting
group for ribonucleosides and its removal through
a decarboxylative
Supporting information for this article is given via a link at the end of
the document.
elimination reaction. Abbreviations: B, uracil-yl (a), cytosin-1-yl (b), adenin-9-yl
(c) or guanin-9-yl (d); TBAF, tetra-n-butylammonium fluoride; TBACl, tetra-n-
butylammonium chloride.
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European Journal of Organic Chemistry
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COMMUNICATION
tetra-n-butylammonium fluoride (TBAF) or chloride (TBACl) in
dry DMSO (Scheme 1) resulted in a clean decarboxylation of 4a
with the concomitant formation of MeCN and release of
formaldehyde to provide uridine (5a) in quantitative yield based
on RP-HPLC analysis of the reaction presented in Figure 1.
Figure 1. RP-HPLC profiles demonstrating the sequential conversion of the
ribonucleoside 2a → 3a/4a → 5a. (A) silica gel purified 2’-O-protected uridine.
(B) de-esterified 2’-O-protected uridine. (C) 2’-O-deprotected uridine. (D)
commercial sample of uridine. Chromatographic conditions are described in
the Materials and Methods section of the Supporting Information.
Although other salts including tetra-n-butylammonium
acetate (TBAOAc) or tetra-n-butylammonium cyanide (TBACN)
could be used in various solvents (e.g., MeCN, MeOH or H2O)
for this purpose, the kinetics of the decarboxylation reaction
were somewhat slower. The mechanistic features of this novel
2’-O-decarboxylative elimination reaction has been scrutinized
by 13C-NMR spectroscopy. Figure 2A displays the 13C-NMR
spectrum of de-esterified 2’-O-protected uridine (3a).
Decarboxylation of 3a/4a has been carried out using ammonium
fluoride, instead of TBAF, in order to prevent interference from
the 13C-NMR signals associated with the tetra-n-butylammonium
cation that would otherwise obscure the diagnostic 13C-signal
shifts resulting from the decarboxylative elimination of MeCN. As
shown in Figure 2B, the signal (11.93 ppm) corresponding to the
methyl group of 3a in Figure 2A, has now shifted to 1.63 ppm;
this signal corresponds to the primary carbon of MeCN.
Consistent with this assessment is the presence of a new signal
at ~118 ppm corresponding to the quaternary carbon of MeCN.
The formation of acetonitrile is further demonstrated by spiking
the NMR sample with commercial MeCN. Figure 2C reveals a
significant increase in the intensity of both 13C signals
corresponding to MeCN, thereby convincingly supporting the
proposed mechanistic pathway taking place during the cleavage
of the iminooxymethyl propanoate group protecting the 2’-
hydroxyl of uridine. With the aim of assessing the generality of
our proposed approach to the protection of ribonucleosides as
2’-O-iminooxymethyl propanoic acid ethyl esters, the 2’-O-
amino-oxymethylribonucleosides 1b-d[13,14] have also been
converted to 2b-d under conditions similar to those employed for
Figure 2. 13C-NMR analysis of the decarboxylative elimination of acetonitrile
from 3a/4a in [D6]DMSO. (A) Spectrum of de-esterified 2’-O-protected uridine
3a. (B) Spectrum of fully 2’-O-deprotected 3a/4a. (C) Spectrum of fully 2’-O-
deprotected 3a/4a that is spiked with commercial acetonitrile.
the preparation of 2a (see experimental details in the Supporting
Information). Treatment of 2b-d with 0.1 M NaOH at 25 °C for 1
h
provides after neutralization with AcOH, the 2’-O-
iminooxymethyl propanoate salt 3b-d in yields similar to that
obtained for 3a. TBAF or TBACl is added to a solution of 3b-d in
dry DMSO; the solution is then heated at 55 °C to induce
decarboxylation of 3b-d over a period of 1 h. Similar to the
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European Journal of Organic Chemistry
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COMMUNICATION
decarboxylation of 3a under those conditions, the concurrent
formation of MeCN and formaldehyde occurred and resulted in
the production of cytidine, adenosine and guanosine in
quantitative yields based on RP-HPLC analysis of the
decarboxylative elimination reactions displayed in Figures S1,
S2 and S3.
snake venom phosphodiesterase and alkaline phosphatase
cleanly led to the production of 3a and thymidine (Figure 5A),
whereas the enzymatic hydrolysis of crude de-esterified 2’-O-
protected (Up)20dT revealed, after decarboxylation, only the
presence of uridine and thymidine (Figure 5B), thereby
indicating complete cleavage of the 2’-O-protective groups from
the chimeric RNA sequence.
Our findings show that an iminooxymethyl propanoic acid
ethyl ester can protect the 2’-hydroxyl of all four ribonucleosides
and be quantitatively removed when needed. In the context of
solid-phase RNA synthesis, it is critically important to assess the
adequacy of the 2’-O-iminooxymethyl propanoate protection/de-
protection process for this purpose. Thus, the solid-phase
synthesis of a chimeric polyuridylic acid (Up)20dT served as a
preliminary and simplistic model for such an assessment. First,
the 5’-O-protection of the ribonucleoside 2a upon reaction with
4,4’-dimethoxytrityl chloride in dry pyridine produced 6 (Scheme
2) in a yield of 90% after silica gel purification. The reaction of 6
with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite in the
presence
of
triethylamine
gives
the
ribonucleoside
phosphoramidite 7 in a post-purification yield of 88%.
Figure 3. RP-HPLC analysis of unpurified and desalted chimeric RNA
sequences. (A) Chromatogram of de-esterified 2’-O-protected (Up)20dT. (B)
Chromatogram of fully 2’-O-deprotected (Up)20dT. (C) Chromatogram of fully
2’-O-deprotected (Up)20dT control sequence. Abbreviations: U, uracil-1-yl; T,
thymin-1-yl. Chromatographic conditions are defined in the Materials and
Methods section of the Supporting Information.
Scheme 2. Synthesis of the ribonucleoside phosphoramidite 7. Experimental
details are provided in the Supporting Information. Abbreviations: DMTrCl,
4,4’-dimethoxytrityl chloride; Ura, uracil-1-yl; 2-CE-DIPCP, 2-cyanoethyl-N,N-
diisopropylchlorophosphoramidite.
The coupling kinetics and efficiency of the ribonucleoside
phosphoramidite 7 were evaluated through the solid-phase
synthesis of (Up)20dT. An identical RNA control sequence was
also prepared using
a
commercial uridine 2’-O-(tert-
butyldimethylsilyl) phosphoramidite[16] with the purpose of
comparing the quality of the RNA sequences produced from
each phosphoramidite monomer. Upon completion of the solid-
phase RNA syntheses, the RNA sequence constructed from the
use of 7 was subjected to treatment with 0.5 M tetra-n-
butylammonium hydroxide for 3 h at 25 °C to concurrently de-
esterify the 2’-O-protecting groups, remove the 2-cyanoethyl
phosphate protective groups and release the RNA sequence
from the solid support. The basic nucleic acid solution was then
neutralized by adding a four-molar equivalent of glacial acetic
acid and evaporated to dryness. Without isolation, the crude
RNA sequence was dissolved in a 0.5 M solution of TBAF or
TBACl in dry DMSO and heated at 55 °C for 3 h to induce
decarboxylation of the 2’-O-iminooxypropanoate groups. The
control RNA sequence was fully deprotected and released from
the support under published conditions.[16,17a,b] Analytical
samples of the unpurified and desalted RNA sequences were
analysed by RP-HPLC and polyacrylamide gel electrophoresis
Figure 4. PAGE analysis of unpurified and desalted chimeric RNA sequences.
Lane A: De-esterified 2’-O-protected (Up)20dT. Lane B: Fully 2’-O-deprotected
(Up)20dT. Lane
C Fully 2’-O-deprotected (Up)20dT control sequence.
Bromophenol blue is used as a marker and appears as a large band at the
bottom of the gel. Electrophoretic conditions are reported in the Materials and
Methods section of the Supporting Information.
(PAGE) under denaturing conditions (Figures
respectively). Moreover, enzymatic hydrolysis of the crude de-
esterified 2’-O-protected (Up)20dT (Figure 3A) catalyzed by
3 and 4,
Although the iminooxymethyl propanoic acid ethyl ester function
has been demonstrated to be generally applicable to the 2’-O-
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European Journal of Organic Chemistry
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COMMUNICATION
protection/deprotection all the four canonical ribonucleosides,
the solid-phase synthesis of RNA sequences carrying all four 2’-
O-protected ribonucleoside is beyond the scope of this
communication and will be dealt with in the near future.
Keywords: nucleosides • 2’-hydroxy protection • protected
protecting group • 2’-O-iminooxymethyl propanoate •
decarboxylative elimination reaction
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Figure 5. RP-HPLC analysis of SVP/BAP hydrolysates. (A) Chromatogram of
unpurified, de-esterified and desalted 2’-O-protected (Up)20dT digest. (B)
Chromatogram of unpurified and desalted 2’-O-deprotected (Up)20dT digest.
(C) Chromatogram of unpurified 2’-O-deprotected (Up)20dT control sequence
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panoic acid ethyl esters, as a novel class of protected protecting
groups,[18] for the 2’-hydroxy protection of ribonucleosides is
unprecedented in that the removal of these protecting groups led
to positive entropic changes through the release of carbon
dioxide, acetonitrile, and formaldehyde. This entropically-driven
process ensures a quantitative intramolecular cleavage of the 2’-
O-protective group under mild and neutral conditions. Given the
notorious complexity of protecting and deprotecting the 2’-
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Experimental Section
Materials and Methods, full experimental procedures and
1
characterization data including H, 13C, 31P NMR and mass spectra of all
new compounds are provided in the Supporting information of this article.
Acknowledgements
This work was supported through FDA intramural funds.
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European Journal of Organic Chemistry
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COMMUNICATION
Layout 2:
COMMUNICATION
Dr.Jacek Cieślak, Dr. Andrzej
Grajkowski, Dr. Cristina Ausίn, and Dr.
Serge L. Beaucage*
2’-O-Aminooxymethylribonucleosides react with ethyl pyruvate to provide, after
saponification, 2’-O-iminooxymethyl propanoate derivatives. Deprotection of the 2’-
hydroxy protecting group to native ribonucleosides is demonstrated through of a
tetraalkylammonium salt-mediated decarboyylative elimination reaction,
Protection of the 2’-Hydroxy Function
of Ribonucleosides as an
Iminooxymethyl Propanoate and its
2’-O-Deprotection via an
Intramolecular Decarboxylative
Elimination Process
Key topic: Decarboxylative
Elimination Reaction
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